US2025234846A1PendingUtilityA1
Genetically modified non-human mammals, preparation methods therefor, and applications thereof
Assignee: RENGENE BIOTECHNOLOGY CO LTDPriority: Jan 19, 2024Filed: Jan 17, 2025Published: Jul 24, 2025
Est. expiryJan 19, 2044(~17.5 yrs left)· nominal 20-yr term from priority
C12N 15/907C12N 15/11C12N 9/22C07K 16/00A01K 2267/01A01K 2227/105A01K 2217/075A01K 2217/052A01K 2207/15A01K 67/0278C12N 2310/20C07K 2317/569C07K 2317/10C07K 2317/21C12N 2015/8518C12N 15/8509C12N 15/113A01K 2217/072C07K 2317/24C12N 2510/00C12N 5/0635C12N 5/0606A01K 67/0276
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Claims
Abstract
The present invention provides a method for preparing a genetically modified non-human mammal for producing a humanized antibody as well as use thereof, which method comprises: (1) bringing about a disruption of an endogenous heavy chain immunoglobulin gene loci in a non-human mammal; and (2) introducing a human IGHV gene, a human IGHD gene, a human IGHJ gene as well as an endogenous IgHG2c gene, IgHE gene, IgHA gene, and LCR region of the non-human mammal into the non-human mammal obtained in step (1). The non-human mammal prepared according to the method of the present invention, after antigen immunization, enables efficiently producing a humanized whole antibody or single heavy chain antibody or nanobody with high immune titer.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A method for preparing a genetically modified non-human mammal, which comprises the following steps:
bringing about a disruption of an endogenous heavy chain immunoglobulin gene loci in a non-human mammal.
2 . The method for preparing a genetically modified non-human mammal according to claim 1 , wherein the non-human mammal is a mouse, and the disruption of the endogenous heavy chain immunoglobulin gene loci include deletions of the following gene fragments:
a CH1 fragment of mouse antibody gene heavy chain IgHM, mouse antibody gene light chain Igκc, and a fragment between IgLc2 and IgLc1 of mouse antibody gene light chain Igλ.
3 . The method for preparing a genetically modified non-human mammal according to claim 2 , wherein the deletions of the gene fragments are performed through CRISPR-Cas9 gene editing technique;
wherein the sgRNA for deleting the CH1 fragment of mouse antibody gene heavy chain IgHM comprises a sgRNA as shown in SEQ ID NO: 1 and a sgRNA as shown in SEQ ID NO: 2; and/or, the sgRNA for deleting the mouse antibody gene light chain Igκc comprises a sgRNA as shown in SEQ ID NO: 3; and/or, the sgRNA for deleting the fragment between IgLc2 and IgLc1 of mouse antibody gene light chain Ig, comprises a sgRNA as shown in SEQ ID NO: 4 and a sgRNA as shown in SEQ ID NO: 5.
4 . The method for preparing a genetically modified non-human mammal according to claim 1 , wherein the non-human mammal is a mouse, and the disruption of the endogenous heavy chain immunoglobulin gene loci include deletions of the following gene fragments:
a fragment between IgHM and IgHA-CH1 of mouse antibody gene heavy chain, mouse antibody gene light chain Igκc, and a fragment between IgLc2 and IgLc1 of mouse antibody gene light chain Igλ.
5 . The method for preparing a genetically modified non-human mammal according to claim 4 , wherein the deletions of the gene fragments are performed through CRISPR-Cas9 gene editing technique;
wherein the sgRNA for deleting the fragment between IgHM and IgHA-CH1 of mouse antibody gene heavy chain comprises a sgRNA as shown in SEQ ID NO: 1 and a sgRNA as shown in SEQ ID NO: 6; and/or, the sgRNA for deleting the mouse antibody gene light chain Igκc comprises a sgRNA as shown in SEQ ID NO: 3; and/or the sgRNA for deleting a fragment between IgLc2 and IgLc1 of mouse antibody gene light chain Ig, comprises a sgRNA as shown in SEQ ID NO: 4 and a sgRNA as shown in SEQ ID NO: 5.
6 . A method for preparing a genetically modified non-human mammal, which comprises the following steps:
(1) preparing a genetically modified non-human mammal I according to the method of claim 1 ; and (2) introducing a human IGHV gene, a human IGHD gene, a human IGHJ gene as well as an endogenous IgHG2c gene, IgHE gene, IgHA gene, and LCR region of the non-human mammal into the genetically modified non-human mammal I obtained in the step (1).
7 . The method for preparing a genetically modified non-human mammal according to claim 6 , wherein the non-human mammal is a mouse, and in the step (2),
the human IGHV gene includes at least the following 18 human IGHV genes: hIGHV4-59, hIGHV3-53, hIGHV5-51, hIGHV3-49, hIGHV3-48, hIGHV1-46, hIGHV3-43, hIGHV4-39, hIGHV3-33, hIGHV4-30-2, hIGHV1-24, hIGHV3-23, hIGHV3-21, hIGHV3-11, hIGHV3-7, hIGHV4-4, hIGHV1-2, hIGHV6-1; optionally, the human IGHV gene further include one or several or all of the human IGHV genes selected from: hIGHV1-58, hIGHV1-45, hIGHV3-35, hIGHV4-28, hIGHV2-26, hIGHV3-20, hIGHV1-18, hIGHV3-15, hIGHV3-13, hIGHV5-10-1, hIGHV3-64D, hIGHV2-5, hIGHV7-4-1, hIGHV1-3; and further optionally, the human IGHV gene further include one or several or all of the human IGHV genes selected from: hIGHV3-60, hIGHV3-57, hIGHV7-56, hIGHV4-55, hIGHV3-54, hIGHV3-52, hIGHV3-50, hIGHV3-47, hIGHV3-42, hIGHV3-41, hIGHV3-38, hIGHV3-37, hIGHV3-36, hIGHV7-34-1, hIGHV4-34, hIGHV3-33-2, hIGHV3-32, hIGHV3-30-2, hIGHV3-30, hIGHV3-29, hIGHV7-27, hIGHV3-25, hIGHV3-22, hIGHV3-19, hIGHV1-17, hIGHV3-16, hIGHV1-14, hIGHV1-12, hIGHV3-6; and/or, the human IGHD gene includes at least the following 15 human IGHD genes: IGHD1-1, IGHD3-3, IGHD6-6, IGHD1-7, IGHD3-10, IGHD6-13, IGHD1-14, IGHD2-15, IGHD3-16, IGHD5-18, IGHD6-19, IGHD1-20, IGHD3-22, IGHD1-26, IGHD7-27; optionally, the human IGHD gene further includes one or several or all of the human IGHD genes selected from: IGHD2-2, IGHD2-8, IGHD3-9, IGHD3-10, IGHD4-11, IGHD5-12, IGHD3-16, IGHD4-17, IGHD2-21, IGHD4-23, IGHD5-24, IGHD6-25; and further optionally, the human IGHD gene further includes one or several or all of the human IGHD genes selected from: IGHD3-3, IGHD3-10, IGHD3-16, IGHD5-18; and/or, the human IGHJ gene includes all the human IGHJ genes; and/or, the endogenous IgHG2c gene of the non-human mammal is the endogenous complete IgHG2c gene, or the endogenous IgHG2c gene segment without the CH1 domain.
8 . The method for preparing a genetically modified non-human mammal according to claim 7 , wherein in the step (2), the human IGHV gene, the human IGHD gene, the human IGHJ gene as well as the endogenous IgHG2c gene, IgHE gene, IgHA gene, and LCR region of the non-human mammal are introduced by the following method:
introducing the following materials into the mouse obtained in the step (1): (I) n BAC clones that comprise all of the human IGHV genes, the human IGHD genes, and the human IGHJ genes, in total; wherein n is an integer between 3 and 8; each of the n BAC clones has a 5 kb to 50 kb of gene fragment at the beginning or the end thereof, and for every two adjacent BAC clones, the 5 kb to 50 kb of gene fragment at the end of one BAC clone is homologous to that at the beginning of the other BAC clone, so that they can be interconnected with each other through the homologous gene sequences; and (II) one or more BAC clones comprising the endogenous IgHG2c gene, IgHE gene, IgHA gene, and LCR region of the non-human mammal.
9 . The method for preparing a genetically modified non-human mammal according to claim 8 , wherein n is an integer between 4 and 7.
10 . The method for preparing a genetically modified non-human mammal according to claim 8 , wherein in the step (2), the human IGHV genes, the human IGHD genes, the human IGHJ genes as well as the endogenous IgHG2c gene, IgHE gene, IgHA gene, and LCR region of the non-human mammal are introduced by the following method:
introducing the following 6 BAC clones into the mouse obtained in step (1): (i) 4 BAC clones containing all the human IGHV genes, (ii) 1 BAC clone containing all the human IGHD genes and all the human IGHJ genes, and (iii) 1 BAC clone containing the endogenous IgHG2c gene, IgHE gene, IgHA gene and LCR region of the non-human mammal; wherein for BAC clones in (i), and for BAC clones in (i) and (ii), each BAC clone has a 5 kb to 50 kb of gene fragment at the beginning or the end thereof, and for every two adjacent BAC clones, the 5 kb to 50 kb of gene fragment at the end of one BAC clone is homologous to that at the beginning of the other BAC clone, so that they can be interconnected with each other through the homologous gene sequences.
11 . The method for preparing a genetically modified non-human mammal according to claim 10 , wherein for BAC clones in (i), and for BAC clones in (i) and (ii), each BAC clone has a 5 kb to 20 kb of gene fragment at the beginning or the end thereof, and for every two adjacent BAC clones, the 5 kb to 20 kb of gene fragment at the end of one BAC clone is homologous to that at the beginning of the other BAC clone, so that they can be interconnected with each other through the homologous gene sequences.
12 . The method for preparing a genetically modified non-human mammal according to claim 10 , wherein the genes contained in the 6 BAC clones are shown in the table below:
BAC
Clones No.
CH17-268I9
CTD-3054M17
CTD-2548B8-CZ
RP11-965B13
CH17-185P21-CZ
RP23-351J19 -CZ
Important
hIGHV3-60
hIGHV3-41
hIGHV3-23
hIGHV3-11
hIGHV1-2
mIgHG2c-CH1
sequence
hIGHV4-59
hIGHV4-39
hIGHV3-22
hIGHV5-10-1
hIGHV6-1
mIgHE
contained
hIGHV1-58
hIGHV3-38
hIGHV3-21
hIGHV3-64D
hIGHD1-1
mIgHA
IGHV3-57
hIGHV3-37
hIGHV3-20
hIGHV3-7
hIGHD2-2
mLCR
hIGHV7-56
hIGHV3-36
hIGHV3-19
hIGHV3-6
hIGHD3-3
hIGHV4-55
hIGHV3-35
hIGHV1-18
hIGHV2-5
hIGHD4-4
hIGHV3-54
hIGHV7-34-1
hIGHV1-17
hIGHV7-4-1
hIGHD5-5
hIGHV3-53
hIGHV4-34
hIGHV3-16
hIGHV4-4
hIGHD6-6
hIGHV3-52
hIGHV3-33-2
hIGHV3-15
hIGHV1-3
hIGHD1-7
hIGHV5-51
hIGHV3-33
hIGHV1-14
hIGHV1-2
hIGHD2-8
hIGHV3-50
hIGHV3-32
hIGHV3-13
hIGHV6-1
hIGHD3-9
hIGHV3-49
hIGHV4-30-2
hIGHV1-12
hIGHD3-10
hIGHV3-48
hIGHV3-30-2
hIGHV3-11
hIGHD4-11
hIGHV3-47
IGHV3-30
hIGHD5-12
hIGHV1-46
hIGHV3-29
hIGHD6-13
hIGHV1-45
hIGHV4-28
hIGHD1-14
hIGHV3-43
hIGHV7-27
hIGHD2-15
hIGHV3-42
hIGHV2-26
hIGHD3-16
hIGHV3-41
hIGHV3-25
hIGHD4-17
hIGHV4-39
hIGHV1-24
hIGHD5-18
hIGHV3-38
hIGHV3-23
hIGHD6-19
hIGHV3-37
hIGHV3-22
hIGHD1-20
hIGHD2-21
hIGHD3-22
hIGHD4-23
hIGHD5-24
hIGHD6-25
hIGHD1-26
hIGHD7-27
hIGHJ1P
hIGHJ1
hIGHJ2
hIGHJ2P
hIGHJ3
hIGHJ4
hIGHJ5
hIGHJ3P
hIGHJ6
wherein “mIgHG2c-CH1” represented the mIgHG2c gene sequence without the CH1 domain.
13 . The method for preparing a genetically modified non-human mammal according to claim 10 , wherein the human IGHV genes, the human IGHD genes, and the human IGHJ genes are operatively linked to each other and subjected to VDJ-rearrangement, and the operatively linked and/or VDJ-rearranged human IGHV genes, the human IGHD genes, and the human IGHJ genes, are operatively linked to the endogenous IgHG2c gene, IgHE gene, IgHA gene, and LCR region of the non-human mammal;
and/or, there is a Switch region of the endogenous IgHM of the non-human mammal between the human IGHJ gene and the endogenous IgHG2c gene of the non-human mammal.
14 . A method for preparing a humanized whole antibody or single heavy chain antibody or nanobody that specifically binds to an antigen, comprising:
(1) the genetically modified non-human mammal prepared by the method of claim 6 is exposed to an antigen; (2) B cells are collected from the non-human mammal obtained in the step (1), RNA is extracted and then reverse transcribed into cDNA, and antibody gene fragments are amplified using the cDNA as the template, and then are cloned into a phage display vector; (3) the phage vector obtained in the step (2) is used to express the target antibody; specifically, phages are subjected to panning to enrich for those expressing the target antibody, which are then induced to express the target antibody, i.e. a humanized whole antibody or single heavy chain antibody; and Optionally, (4) the variable region fragments of the obtained single heavy chain antibody are obtained by cloning to obtain a humanized nanobody.
15 . A method for preparing a humanized whole antibody or single heavy chain antibody or nanobody that specifically binds to an antigen, comprising:
(1) the genetically modified non-human mammal prepared by the method of claim 6 is exposed to an antigen, and then B cells are collected; (2) nucleic acids encoding the immunoglobulin heavy chain variable region and optional the light chain variable region in B cells collected in the step (1) are sequenced to obtain the nucleic acid sequences of a heavy chain variable region and a light chain variable region of a humanized monoclonal antibody or the nucleic acid sequence of a heavy chain variable region of a humanized nanobody, (3) according to the sequences obtained in the step (2), a humanized whole antibody or a humanized single heavy chain antibody that specifically binds to the antigen is obtained by expression; and Optionally, (4) the variable region fragments of the obtained single heavy chain antibody are obtained by cloning to obtain a humanized nanobody.
16 . A sgRNA composition, which comprises: sgRNA for deleting CH1 fragment of mouse antibody gene heavy chain IgHM; sgRNA for deleting mouse antibody gene light chain Igκc; and sgRNA for deleting a fragment between IgLc2 and IgLc1 of mouse antibody gene light chain Igλ.
17 . The sgRNA composition of claim 16 , wherein the sgRNA for deleting the CH1 fragment of mouse antibody gene heavy chain IgHM comprises a sgRNA as shown in SEQ ID NO: 1 and a sgRNA as shown in SEQ ID NO: 2; and/or, the sgRNA for deleting the mouse antibody gene light chain Igκc comprises a sgRNA as shown in SEQ ID NO: 3; and/or, the sgRNA for deleting the fragment between IgLc2 and IgLc1 of mouse antibody gene light chain Ig, comprises a sgRNA as shown in SEQ ID NO: 4 and a sgRNA as shown in SEQ ID NO: 5.
18 . A sgRNA composition, which comprises: sgRNA for deleting a fragment between IgHM and IgHA-CH1 of mouse antibody gene heavy chain; sgRNA for deleting mouse antibody gene light chain Igκc; and sgRNA for deleting a fragment between IgLc2 and IgLc1 of mouse antibody gene light chain Igλ.
19 . The sgRNA composition of claim 18 , wherein the sgRNA for deleting the fragment between IgHM and IgHA-CH1 of mouse antibody gene heavy chain comprises a sgRNA as shown in SEQ ID NO:1 and a sgRNA as shown in SEQ ID NO:6; and/or, the sgRNA for deleting the mouse antibody gene light chain Igκc comprises a sgRNA as shown in SEQ ID NO: 3; and/or, the sgRNA for deleting the fragment between IgLc2 and IgLc1 of mouse antibody gene light chain Ig, comprises a sgRNA as shown in SEQ ID NO:4 and a sgRNA as shown in SEQ ID NO:5.
20 . A genetically modified non-human mammal, which comprises a disruption in an endogenous heavy chain immunoglobulin gene loci, and the endogenous heavy chain immunoglobulin gene loci comprises a human IGHV gene, a human IGHD gene, a human IGHJ gene as well as an endogenous IgHG2c gene, IgHE gene, IgHA gene, and LCR region of the non-human mammal.
21 . The genetically modified non-human mammal according to claim 20 , wherein the disruption in the endogenous heavy chain immunoglobulin gene loci includes deletions of the following gene fragments:
a CH1 fragment of mouse antibody gene heavy chain IgHM, mouse antibody gene light chain Igκc, and a fragment between IgLc2 and IgLc1 of mouse antibody gene light chain Igλ; and/or, the human IGHV gene includes at least the following 18 human IGHV genes: hIGHV4-59, hIGHV3-53, hIGHV5-51, hIGHV3-49, hIGHV3-48, hIGHV1-46, hIGHV3-43, hIGHV4-39, hIGHV3-33, hIGHV4-30-2, hIGHV1-24, hIGHV3-23, hIGHV3-21, hIGHV3-11, hIGHV3-7, hIGHV4-4, hIGHV1-2, and hIGHV6-1; preferably, the human IGHV gene further includes one or several or all of the human IGHV genes selected from: hIGHV1-58, hIGHV1-45, hIGHV3-35, hIGHV4-28, hIGHV2-26, hIGHV3-20, hIGHV1-18, hIGHV3-15, hIGHV3-13, hIGHV5-10-1, hIGHV3-64D, hIGHV2-5, hIGHV7-4-1, and hIGHV1-3; further preferably, the human IGHV gene further includes one or several or all of the human IGHV genes selected from: hIGHV3-60, hIGHV3-57, hIGHV7-56, hIGHV4-55, hIGHV3-54, hIGHV3-52, hIGHV3-50, hIGHV3-47, hIGHV3-42, hIGHV3-41, hIGHV3-38, hIGHV3-37, hIGHV3-36, hIGHV7-34-1, hIGHV4-34, hIGHV3-33-2, hIGHV3-32, hIGHV3-30-2, hIGHV3-30, hIGHV3-29, hIGHV7-27, hIGHV3-25, hIGHV3-22, hIGHV3-19, hIGHV1-17, hIGHV3-16, hIGHV1-14, hIGHV1-12, and hIGHV3-6; and/or, the human IGHD gene includes at least the following 15 human IGHD genes: IGHD1-1, IGHD3-3, IGHD6-6, IGHD1-7, IGHD3-10, IGHD6-13, IGHD1-14, IGHD2-15, IGHD3-16, IGHD5-18, IGHD6-19, IGHD1-20, IGHD3-22, IGHD1-26, and IGHD7-27; preferably, the human IGHD gene further includes one or several or all of the human IGHD genes selected from: IGHD2-2, IGHD2-8, IGHD3-9, IGHD3-10, IGHD4-11, IGHD5-12, IGHD3-16, IGHD4-17, IGHD2-21, IGHD4-23, IGHD5-24, and IGHD6-25; further preferably, the human IGHD gene further includes one or several or all of the human IGHD genes selected from: IGHD3-3, IGHD3-10, IGHD3-16, and IGHD5-18; and/or, the human IGHJ gene includes all the human IGHJ genes; and/or, the endogenous IgHG2c gene of the non-human mammal is the complete endogenous IgHG2c gene, or the endogenous IgHG2c gene fragment without the CH1 domain; preferably, the human IGHV genes, the human IGHD genes, and the human IGHJ genes are operatively linked to each other and subjected to VDJ-rearrangement; further preferably, the operatively linked and/or VDJ-rearranged human IGHV genes, human IGHD genes, and human IGHJ genes, are operatively linked to the endogenous IgHG2c gene, IgHE gene, IgHA gene, and LCR region of the non-human mammal; preferably, there is a Switch region of the endogenous IgHM of the non-human mammal between the human IGHJ gene and the endogenous IgHG2c gene of the non-human mammal; most preferably, the endogenous heavy chain immunoglobulin gene loci of the non-human mammal comprises all the genes shown in Table 13.
22 . The genetically modified non-human mammal according to claim 20 , wherein the disruption in the endogenous heavy chain immunoglobulin gene loci includes deletions of the following gene fragments:
a fragment between IgHM and IgHA-CH1 of mouse antibody gene heavy chain, mouse antibody gene light chain Igκc, and a fragment between IgLc2 and IgLc1 of mouse antibody gene light chain Igλ; and/or, the human IGHV gene includes at least the following 18 human IGHV genes: hIGHV4-59, hIGHV3-53, hIGHV5-51, hIGHV3-49, hIGHV3-48, hIGHV1-46, hIGHV3-43, hIGHV4-39, hIGHV3-33, hIGHV4-30-2, hIGHV1-24, hIGHV3-23, hIGHV3-21, hIGHV3-11, hIGHV3-7, hIGHV4-4, hIGHV1-2, and hIGHV6-1; preferably, the human IGHV gene further includes one or several or all of the human IGHV genes selected from: hIGHV1-58, hIGHV1-45, hIGHV3-35, hIGHV4-28, hIGHV2-26, hIGHV3-20, hIGHV1-18, hIGHV3-15, hIGHV3-13, hIGHV5-10-1, hIGHV3-64D, hIGHV2-5, hIGHV7-4-1, and hIGHV1-3; further preferably, the human IGHV gene further includes one or several or all of the human IGHV genes selected from: hIGHV3-60, hIGHV3-57, hIGHV7-56, hIGHV4-55, hIGHV3-54, hIGHV3-52, hIGHV3-50, hIGHV3-47, hIGHV3-42, hIGHV3-41, hIGHV3-38, hIGHV3-37, hIGHV3-36, hIGHV7-34-1, hIGHV4-34, hIGHV3-33-2, hIGHV3-32, hIGHV3-30-2, hIGHV3-30, hIGHV3-29, hIGHV7-27, hIGHV3-25, hIGHV3-22, hIGHV3-19, hIGHV1-17, hIGHV3-16, hIGHV1-14, hIGHV1-12, and hIGHV3-6; and/or, the human IGHD gene includes at least the following 15 human IGHD genes: IGHD1-1, IGHD3-3, IGHD6-6, IGHD1-7, IGHD3-10, IGHD6-13, IGHD1-14, IGHD2-15, IGHD3-16, IGHD5-18, IGHD6-19, IGHD1-20, IGHD3-22, IGHD1-26, and IGHD7-27; preferably, the human IGHD gene further includes one or several or all of the human IGHD genes selected from: IGHD2-2, IGHD2-8, IGHD3-9, IGHD3-10, IGHD4-11, IGHD5-12, IGHD3-16, IGHD4-17, IGHD2-21, IGHD4-23, IGHD5-24, and IGHD6-25; further preferably, the human IGHD gene further includes one or several or all of the human IGHD genes selected from: IGHD3-3, IGHD3-10, IGHD3-16, and IGHD5-18; and/or, the human IGHJ gene includes all the human IGHJ genes; and/or, the endogenous IgHG2c gene of the non-human mammal is the complete endogenous IgHG2c gene, or the endogenous IgHG2c gene fragment without the CH1 domain; preferably, the human IGHV genes, the human IGHD genes, and the human IGHJ genes are operatively linked to each other and subjected to VDJ-rearrangement; further preferably, the operatively linked and/or VDJ-rearranged human IGHV genes, human IGHD genes, and human IGHJ genes, are operatively linked to the endogenous IgHG2c gene, IgHE gene, IgHA gene, and LCR region of the non-human mammal; preferably, there is a Switch region of the endogenous IgHM of the non-human mammal between the human IGHJ gene and the endogenous IgHG2c gene of the non-human mammal; most preferably, the endogenous heavy chain immunoglobulin gene loci of the non-human mammal comprises all the genes shown in Table 13.Join the waitlist — get patent alerts
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